Cellulosic biomass is a significant renewable resource for the generation of sugars. Fermentation of these sugars can yield numerous end-products such as fuels and chemicals, which are currently derived from petroleum. While the fermentation of sugars to fuels such as ethanol is relatively straightforward, the hydrolytic conversion of cellulosic biomass to fermentable sugars such as glucose is difficult because of the crystalline structure of cellulose and its close association with lignin (Ladisch, et al., 1983 Enzyme Microb. Technol. 5:82). Pretreatment, by means, including but not limited to, mechanical and solvent means, increases the susceptibility of cellulose to hydrolysis. Pretreatment may be followed by the enzymatic conversion of cellulose to glucose, cellobiose, cello-oligosaccharides and the like, using enzymes that specialize in breaking down the β-1-4 glycosidic bonds of cellulose. These enzymes are collectively referred to as “cellulases”.
Cellulases may be divided into three major sub-categories of enzymes: 1,4-β-D-glucan glucanohydrolases (“endoglucanases” or “EGs”); 1,4-β-D-glucan cellobiohydrolases (“exoglucanases”, “cellobiohydrolases”, or “CBHs”); and β-D-glucoside-glucohydrolases (“β-glucosidases”, “cellobiases” or “BGs”). Endoglucanases randomly attack the interior parts, and mainly the amorphous regions of cellulose, mostly yielding glucose, cellobiose, and cellotriose. Exoglucanases incrementally shorten the glucan molecules by binding to the glucan ends and releasing mainly cellobiose units from the ends of the cellulose polymer. β-glucosidases split the cellobiose, a water-soluble β-1,4-linked dimer of glucose, into two units of glucose. The cellulase enzyme classification can be further expanded to include multiple components within each cellulase classification. For example, numerous EGs, CBHs and BGs have been isolated from a variety of organisms such as Trichoderma reesei and Humicola insolens. It is known that Trichoderma reesei contain at least 8 EGS, including EGI, EGII, EGIII, EGIV, EGV, EGVI, EGVII and EGVIII; at least 5 BGs, including BG1, BG2, BG3, BG4, BG5 and at least 2 CBHs (CBH1 and CBH2) (Foreman P. K. J. Biol. Chem. 2003, 278:31988-31997).
Most CBHs are multi-domain proteins consisting of a catalytic domain and a cellulose or carbohydrate binding domain (CBD) separated by a linker region. The catalytic domain is responsible for cleavage of the cellulose. The catalytic domain is classified into the glycoside hydrolase family wherein the family members include enzymes having a similar fold and hydrolytic mechanisms. The CBH2s (Cel6) are members of the glycoside hydrolase Family 6. The three dimension structure of a number of CBHs is known. Generally CBH2 enzymes operate on the non-reducing end of a cellulase substrate as compared to CBH1 enzymes. In addition, there are a number of CBH2s which do not include a CBD.
The use of cellulase enzymes in various industrial applications is well known. Cellulases have been used in the treatment of textiles for the purpose of enhancing the cleaning ability of detergent compositions, for use as a softening agent for improving the feel and appearance of cotton fabrics; and for denim finishing (U.S. Pat. No. 5,648,263, U.S. Pat. No. 5,776,757, and Kumar et al., Textile Chemist and Colorist 1997, 29:37-4). Cellulases have also been used in the pulp and paper industry for treating fibers, in the food industry, and as an additive in animal feed. In addition, cellulases have been used in the saccharification process to hydrolyze carbon substrates (including both starch and cellulose), to fermentable sugars.
The production of fermentable sugars from renewable biomass substrates (e.g., lignocellulosic feedstocks) with the sequential or simultaneous production of fuel products and/or other chemical end-products to reduce dependence on fossil fuels has emerged as a worldwide recognized goal.
While cellulase compositions and modified cellulase enzymes have been previously described (US Patent Publication No. 20060205042 and US Patent Publication No. 20080076152) cellulases that exhibit improved performance characteristics such as but not limited to increased thermo-stability, improved stability, improved activity, and the like are of particular interest.